Enteric infections, including typhoid fever, are a major source of morbidity and mortality worldwide. The lack of information regarding the immunological correlates of protection against these organisms in humans has hindered the greatly needed development of new and improved vaccines against these important pathogens. The availability of unique specimens from subjects immunized and/or challenged with wild-type (wt) Salmonella enterlca serovar Typhi (S. Typhi), as well as specimens from subjects immunized with the licensed Ty21a oral typhoid vaccine in conjunction with novel technologies and instrumentation (e.g., mass flow cytometry) provide an unprecedented opportunity to investigate the key immunological responses involved in protection at both the systemic and mucosal levels. Our overarching hypothesis is that the delicate homeostasis between the effector and regulatory arms of the immune system, both systemically and in the gut mucosa, plays a critical role in protection from typhoid fever in humans. To begin to understand the complex interaction of various cell subsets of the systemic and mucosal immune systems that may play a role in protection against S. Typhi in humans, we will evaluate the following hypotheses: (1) Protection from S. Typhi disease in a human challenge model is determined by the balance of effector and regulatory responses; (2) Protection induced by immunization with attenuated S. Typhi strains is determined by the balance of effector and regulatory responses; (3) Increased levels of mucosa-associated invariant T (MAIT) cell activation and exhaustion following exposure to S. Typhi antigens contributes to the development of typhoid fever; and (4) Oral vaccination with Ty21a elicits immune responses at the mucosal and systemic levels that differ in magnitude and characteristics. Furthermore, we will integrate the immunological data generated in this project with the composition and functional properties of the gut microbiota (Research Project 2) and the mechanistic studies of mediators of protection at the mucosal level in an in vitro 3-D bioengineered human intestinal tissue model (Research Project 3). These studies will address critical gaps in knowledge and provide the most comprehensive investigation of the correlates of protection against S. Typhi to date which may also be broadly applicable to other enteric pathogens.